Osteoblast differentiation is marked by the sequential activation of many genes encoding the extracellular matrix proteins such as alkaline phosphatase (APase), collagens, proteoglycans, bone sialoprotein (BSP), osteopontin (OPN), and osteocalcin (OC), and hormone receptors such as vitamin D3 and parathyroid hormone. Osteocalcin is expressed in high levels only in mature osteoblasts. Osteoblast cell line representing various stages in the osteoblast lineage are useful in studying osteoblast-specific gene expression. Three unique clonal murine osteosarcoma lines (K8, K14, and K37) were grown in vitro under conditions permissive for mineralization. K8 cells constitutively express BSP, OPN, APase, OC, and Type I collagen at the highest levels as determined by Northern hybridization. Subconfluent K14 cells show expression of the osleogenic phenotype, including OC, with down-regulation after several days in culture. K37 cells are non-osteogenic and used as a control, 1,25-dihydroxyvitami n D3 (1,25-D3) stimulated expression of OC in K14 cells, was weakly inhibitory in K8, and had no effect on K37. In contrast to its variable effect on-OC expression, 1,25-D3 stimulated OPN expression in all cell lines. The osteoblast-specific cis-acting element OSE2 has been shown to present in the mouse OC promoter and to confer activity to a heterologous promoter and to bind atranscription factor, cbfal, present only in nuclear extracts of osteoblasts. Electromobility shift assays (EMSA) revealed the presence of cbfal in nuclear extracts of K8 cells throughout the mineralization period, while K14 showed it present only during early, pre-mineralization conditions. The presence of 1,25-D3 had little influence on cbfal expression. Studies are being conducted to determine the effect of the vitamin D response element on the expression of OPN and OC. The in vitro data, along with previous in vivo data, suggest that K8 are constitutively osteogenic and appear uncoupled from systemic and local tissue regulation. K14 are conditionally ostcogenic, capable of up-regulation of their osteogenic phenotype by such factors as 1,25-D3.